IOSR Journal Of Pharmacy

(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219

www.iosrphr.org Volume 4, Issue 11 (November 2014), PP. 53-64

Clinical, pharmacokinetic and technological aspects of the

hydroxychloroquine sulfate
Leslie Raphael de Moura Ferraz1; Fabiana Lcia Arajo dos Santos2; Pablo de
Atade Ferreira3; Ricardo Tadeu Loureno Maia Junior4; Talita Atanazio Rosa5,
Larissa Arajo Rolim6; Pedro Jos Rolim-Neto7.
1

Ph.D Student, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Brazil

Ph.D Student, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Brazil
3
Ph.D Student, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Brazil
4
Graduate Student, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Brazil
5
MSc. Student, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Brazil
7
Professor, Pharmaceutical Sciences Collegiate, Federal University of Vale do So Francisco, Brazil
8
Professor, Department of Pharmaceutical Sciences, Federal University of Pernambuco, Brazil
2

ABSTRACT : Developed originally as an antimalarial agent, hydroxychloroquine sulfate (HCQS) is often used
as a slow-acting drug in treating disorders of connective tissue. Over the past two decades, several data have
been accumulated on the systemic effects of HCQS, expanding the potential uses of this drug in different
therapeutic classes. The purpose of this article was to conduct a narrative review with qualitative approach on
clinical, pharmacokinetic and technological aspects of HCQS, aiming to gather relevant pieces of information
for the development of new therapeutic approaches to this drug. A search of the literature of scientific
experimental and theoretical studies in the period 1980-2013 was performed. According to the data collected,
among the activities HCQS, there are the indications for the treatment of autoimmune diseases such as lupus
erythematosus and rheumatoid arthritis. Reports also indicate that HCQS improves insulin sensitivity, ability to
reduce thromboembolic events, reduction of lipid levels and treatment for infection by human immunodeficiency
virus. The evidence found out ocular and cutaneous adverse effects and the formation of three chiral active
metabolites, what encourages studies to evaluate the kinetic behavior of HCQS and the intrinsic
physicochemical characteristics of the drug, which is yet poorly described in the literature.

KEYWORDS : Autoimmune Diseases, Chiral Drugs, HIV, Hydroxychloroquine Sulfate .

I.

INTRODUCTION

Initially developed as an antimalarial agent, hydroxychloroquine sulfate (HCQS) - chemically 2-({4[(7-chloroquinolin-4-yl)amino]pentyl}(ethyl)amino)ethan-1-ol - is often used as slow-acting antirheumatic drug
in the treatment of disorders of connective tissue [1-3]. As antimalarial, hydroxychloroquine (HCQ) is
significantly effective against the erythrocytic form of the etiological agents of malaria: Plasmodium
vivax and Plasmodium malariae, and most of Plasmodium falciparum strains. However, recently some
resistance has been observed to chloroquine-resistant Plasmodium falciparum, as well as cases of
resistance against Plasmodium vivax strains. And, despite being used a long time ago in this type of therapy, it is
not known its exact mechanism of action [4-5].
The HCQ has been used also as a secondary drug in the treatment of a variety of chronic diseases. So,
they are administered in conjunction with other agents, resulting in clinical efficacy of diseases such as
rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), discoid lupus (LD), sarcoidosis, Sjgren's
syndrome (SS) and photosensitivity diseases [5].The treatment of autoimmune diseases with anti-malarial have
been common for over half a century. Many long-term studies show better results in terms of organ damage and
overall survival of patients receiving these drugs. Over the past two decades, more data have been accumulated
on the systemic effects of HCQS, expanding the potential uses of this medication in different therapeutic classes
[1].The purpose of this article was to conduct a narrative review with qualitative feature on clinical,
pharmacokinetic and technological aspects of HCQS, aiming to gather relevant information for development of
new therapeutic approaches to this drug.

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Clinical, pharmacokinetic and technological

It was performed a literature search of scientific experimental and theoretical studies in the period of
1980-2013. However, some previous publications of essential scientific relevance about the study topic were
considered. All the sources surveyed integrate scientific articles that address the topic of HCQS, besides
information from official sites and master or PhD thesis.

II.

CLINICAL ASPECTS

The HCQS brings benefits to long term in the treatment of SLE and RA and has become a standard
component of therapy for patients with these diseases. Table 1 provides a systematic overview of the
effectiveness of treatment with HCQS on clinical manifestations of major diseases where the use of the drug is
recommended.
Table 1. Systematic review of evidence of efficacy of HCQS in the treatment of RA and SLE [1].
Illness

Evaluation of efficacy

Type of evidence

Reference

RA

Counting of joint swelling

and pain;

Systematic literature;

[6]

Controlled trial, double-blind;

[7]

Clinical randomized, controlled,

double-blind;

[8]

Articular index;

Joint pain and sensitivity;

SLE

Seizure frequency;

Organ failure;
Survival;

Randomized, controlled, doubleblind, involving withdrawal of

therapy;
Longitudinal cohort study; casecontrol study;
Analysis of prospective cohort;
Analysis of longitudinal cohort;

[9]

[10-11]
[12-13]

Late onset of the disease;

Nested case-control study;

[14]

Cutaneous manifestations;

Analysis of prospective cohort;

[15]

Prevalence of new renal

disease;

Analysis of prospective cohort;

[13]

Analysis of longitudinal cohort;

[16]

Retrospective cohort analysis;

[17]

Glomerulonephritis;
Complete renal remission for
membranous nephropathy.

Reports demonstrated the efficacy of HCQ in different conditions from those which have rheumatic
diseases. Some of these reports are described below [1].The first information relates to diabetes mellitus.
Hypoglycemic conditions in patients treated with HCQ and CQ has being observed. Three small clinical studies,
randomized and controlled, using patients without other rheumatic complications and receiving a relatively high
dose of HCQS (600 mg) reinforced this situation [18].A brief prospective study with obese, non-diabetic and
non-carriers of autoimmune diseases demonstrated an improvement of insulin sensitivity after six weeks of
treatment with HCQ. These results suggest that there is not only a reduction in the incidence of type two

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Clinical, pharmacokinetic and technological

diabetes in patients who are being treated with HCQS for other conditions, as well as in the rate of blood
glucose and insulin resistance.
Probably, this effect of HCQ on the glucose is related to changes in the intra-endosomal pH, which
results in reduced degradation of insulin in human adipose tissue [1]; [19].Another interesting finding is the
ability of HCQ to reduce thromboembolic events. Johnson & Charnley (1979) [20] reported the usefulness of
prophylactic HCQS to avoid significant thrombotic events in post-operative conditions of total hip
arthroplasty. An analysis of prospective cohort demonstrated the protective effect of antimalarial drugs in
development of thrombosis as well as significant improvement of survival. A more recent study - cohort-based
analysis, using 1930 patients with SLE - examined factors that were associated with one or more thrombotic
events, which showed that the use of HCQS was significantly associated with a risk reduced thrombosis,
especially in young patients [12]; [21-24].
Several mechanisms have been proposed for the antithrombotic effects of HCQS. The most probable
appears to be the reduction of platelet aggregation or the inhibition of the release of alpha granule, structures
that have factors which participate in the coagulation cascade. However, these findings are limited to in
vitro studies and the concentration necessary to be able to observe this effect (25 mg / ml) is 200 times higher
than levels reported in the blood of treated patients [15]; [24-25].A reduction in cardiovascular risk, due to
general beneficial effect on the lipid profile when treated with HCQS, has been recognized for decades. Petri
and collaborators (1994) [26], in longitudinal cohort study showed a significant association between treatment
with HCQS, with a dose of 200 mg to 400 mg per day, and lower cholesterol levels. Similar results were
obtained by Rahman and collaborators (1999) [27], emphasizing the recovery of cholesterol levels when in
discontinuation of the therapy. Thus, recent studies have proved reducing lipid levels, total cholesterol and even
low density lipoproteins in patients with RA or SLE, who are treated with the drug in question, for relatively
short periods [28-33].
The mechanisms responsible for changes in the lipid profile with HCQS are not well
elucidated. Studies have postulated that this is due to significantly higher rate of clearance of lipid fractions or
even to upregulation of low-density lipoprotein receptor [34].Perhaps the most unexpected factor is the large
amount of published literature documenting the use of antimalarials, especially HCQS, in the treatment of
infection with the human immunodeficiency virus (HIV). Just over a decade, two controlled clinical trials have
demonstrated the efficacy of HCQS in this illness.The first of these, a placebo-controlled study, which 40
patients with CD4+ T lymphocytes levels greater than 200 cells/mm3 were randomized to receive 800 mg/day of
HCQS or placebo for 8 weeks. The treated group showed a significant decline in levels of viral ribonucleic acid
(RNA), while this value was higher in the group treated with placebo. The percentage of CD4+ T cells remained
stable [35].The second study featured 72 seropositive HIV patients, which were randomized to receive the same
dose of the drug, but this time, instead of placebo, would receive 500 mg/day of zidovudine (ZDV) in a doubleblind scheme. The group treated with antimalarial showed a significant decrease of viral RNA in plasma, after
16 weeks of treatment, maintaining a stable level of CD4+ T cells, and low levels of interleukin-6 (IL-6) and
immunoglobulin G (IgG) after treatment. The group treated with ZDV also had significantly lower viral load,
but there were no changes of great magnitude in serum levels of IL-6 or IgG. Given that high levels of IL-6 are
usually associated with a higher risk of progression of HIV, these results were encouraging with HCQS in terms
of immune modulation, since these are usually favorable and can contribute to the overall effectiveness of
therapy at least in some infected patients [1]; [36].
Paton and Aboulhab (2005) [37] in a recent study using a lower dose of HCQS (200 mg/day),
hydroxyurea and didanosine - reverse transcriptase inhibitor - prove a significant reduction of viral load in about
half of the patients, with levels undetectable at the end of the study, remaining constant the level of CD4 + T
lymphocytes.At first, the idea to fabricate dosage forms containing a relatively inexpensive drug for the therapy
of acquired immunodeficiency syndrome (AIDS) was considered plausible, aiming to use them in less wealthy
countries with high rates of HIV infection. In contrast, the availability of generic drugs at low cost and
government support in such treatment reduced the urgency and concern about the approach. However, the use of

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Clinical, pharmacokinetic and technological

HCQ adjuvant in fixed dose combination with certain drugs for the treatment of AIDS has been widely
discussed [38-39].The antimicrobial mechanism of this action seems to be the alkalinization of acidic
intracellular vesicles, which inhibit the growth of microorganisms.
Studies also suggest that the reduction in the viral load in HIV infection occurs due to disturbance of
post-translational glycosylation of envelope protein of the virus (gp120 protein), resulting in reduced infectivity
of newly produced viral particles [39-41].The risks of the treatment with HCQS are relatively low. Overall, it
correspond to gastrointestinal intolerance (nausea, vomiting, abdominal pain), skin hyperpigmentation,
bleaching of hair, headache, dizziness. However, it remains a specific potential for more serious adverse events
such as retinal damage (retinopathy) and neuromyotoxicity [1]; [42-44].
Many of these adverse effects seem to be due to increased endosomal pH, which changes the
processing of certain proteins and the binding to receptors of biological components. Other studies have
postulated different mechanisms, such as strong links in vitro drug: nucleic acids and inhibition of calcium
signaling in cells lymphoid cells [45-46].Ocular and cutaneous adverse effects of HCQ are possibly phototoxic
reactions. Reports of drug substances that cause phototoxicity, a significant number of them contain chlorine,
such as HCQ. It is suggested that the free radical chloride, formed from breaking the C-Cl bond, is the one
responsible for the damage, since this combines with skin proteins [47-49].For reasons such as these, the use of
antimalarials requires regular medical supervision and monitoring.Ocular toxicity associated with antimalarial
agents was first observed in 1957. In 1959, Hobbs & Sorsby recognized for the first time the retinal toxicity
associated with the use of HCQ [50-51].Studies show that the frequency of retinopathy caused by
accumulation of HCQ ranges from 0.001 to 40%, but millions of people use the medicine for various
purposes. However, ophthalmologic toxicity of this drug is a serious concern, because even after withdrawal of
medication, there is little or no visual recovery, and sometimes there is progression of visual loss [52-56].
The mechanism causing ocular toxicity is not well understood. Antimalarial agents have acute effects
on the metabolism of retinal cells, including photoreceptors. However, it is unclear whether these metabolic
effects are the causes of the slow and chronic damage, in short-term, that characterize the clinical status of
toxicity. HCQ binds to melanin in the retinal pigment epithelium, and this bond concentrate HCQ and contribute
or prolong its toxic effects [57].New data showed that the risk of toxicity increases sharply to 1% after 5 to 7
years of use, or a cumulative dose of 1000 g HCQS, considering that the single dose is generally 400 mg or
calculated by weight dependence. The risk increases with continued use of the drug. Renal or hepatic failure
should be considered, since these are the organs responsible for drug clearance. Age, genetic factors and patients
with maculopathy should also be considered as causal factors for treatment discontinuation. Prolonged treatment
is not recommended in children and in patients with hypersensitivity to 4-aminoquinolines [57].It is
recommended to patients who will undergo the use of antimalarial drugs an initial examination to serve as a
reference point and to discard maculopathy. This is a worsening of retinopathy of genetic nature, which may
prove to be a contraindication to the use of these therapeutic agents. The protocol of annual screening should
begin after 5 (five) years of continuous drug exposure. Tests such as multifocal electroretinography, optical
coherence tomography, background autofluorescence, beyond the usual tests as visual field and fundus
examinations are highly recommended [57].
It is advised that patients should be aware of the risk of toxicity and fundamentals for screening, aiming
to detect, or minimize the impact of the effect. The drugs should be discontinued, if possible, when toxicity is
recognized or strongly suspected. This is a decision to be taken together: patients and their physicians [57].The
use of the drug in question is secure during pregnancy, because the molecule is large and HCQ is not able to
cross the placental barrier. Cross-sectional studies suggest that it is possible to reduce the risk of fetal cardiac
abnormalities in mothers with autoantibodies against antigens that participate in processes that involve RNA
polymerase [58-61].

III.

PHARMACOKINETIC ASPECTS

The HCQS is marketed and administered to patients as racemate, equimolar mixture of two
enantiomers: (+)-HCQ and (-)-HCQ. However, no information is available about the possibility of
stereoselective disposition and pharmacological activities of the isolated enantiomers [2]; [62].Despite this fact,

56

Clinical, pharmacokinetic and technological

many studies seek to measure the concentration of each of the enantiomers of HCQ and its respective
metabolites. McLachlan and collaborators (1993) [3] suggested the existence of the action of one or more
stereoselective processes in the arrangement of the molecule of HCQ in plasma and urine. Such processes are:
chiral inversion, absorption, distribution and renal excretion. Nevertheless,
considering the stability of vicinal groups of chiral center in the molecule, the possibility of chiral
inversion seems remote. The others showed to be stereoselective [2]; [63-66].The high concentrations of R-(-)HCQ in the blood and plasma compared to those seen with (+)-S-HCQ, confirm the existence of stereoselective
processes in the disposition of the drug. It is postulated that the enantiomers (R) is preferably concentrated by
cellular blood compounds, and once set, would leave the (S) enantiomer longer available for
metabolism. However, studies show that this and other stereoselective processes have significant variability
between different individuals [2]; [66-67].There is no information available which ensure the efficacy and
toxicology of each of the enantiomers. It is also unclear which of them is responsible for the antiarthritic
activity. Haberkorn and collaborators (1979) [68] showed limited toxicity data in mice showing that R-(+)-CQisomer is more toxic to possess lethal dose lower than its enantiomer (S). The work also demonstrates that the S(+)-CQ, which has a very similar structure to HCQ, showed to have more potent antimalarial activity that R-(-)CQ, in mice. However, the simple structural similarity does not allow extrapolation of the data mentioned above
for the treatment of RA [64].
The HCQS presents oral and intestinal absorption variables with bioavailability of approximately 74%
and distributed throughout the body with prolonged retention in the eye, liver, skin, lungs, other areas rich in
melanin. In the epidermis, the drug concentration can become 100 to 200-fold higher than plasma levels. In the
erythrocyte, concentration is 2 to 5 times greater than plasma, since HCQ has substantial capacity for protein
binding [44]; [65]; [69].The metabolism of the 4-aminoquinoline derivative is complex and extensive. After
long-term administration, the plasma contains significant levels of HCQ and its three major metabolites, which
still have the chiral center. Therefore, all exist as pairs of enantiomers, eight different substances [70].The
accumulation of the drug and its metabolites should be expected in chronic dosing. The compound has long
plasma half-life, estimated to exceed 40 days, and soon it may reach high concentrations (6000-80000 times
higher than the plasma level). In addition, the plasma half-life increases proportionally with increasing
dose. Current studies are able to measure this pharmacokinetic parameter by HPLC in whole blood [3]; [44];
[69]; [71].
The main route of excretion is renal, with 23-25% of the excreted compound in its unmodified form,
along with the metabolites. Ducharme and collaborators (1995) [72] observed more rapid elimination of the (S)(+)-HCQ enantiomer compared with the (R)- (-)-HCQ, probably due to more rapid hepatic metabolism and
excretion. This result was corroborated by Fieger and collaborators (1993) [73]. The work has also showed that
the metabolites derived from (S) enantiomers represented 80-90% of the dose recovered and urinary metabolites
were not detected in the blood.Unlike what it was stated by Tett & collaborators (1989) [3], although HCQ
overdoses is rarely reported, seven cases were highlighted in a literature review performed by Marquardt and
collaborators (2001) [74]. However, there is no harmonization of treatment of this situation.

IV.

TECHNOLOGICAL ASPECTS

Preformulation studies can be defined as the full investigation of the physico-chemical properties of the
active, alone or combined with other excipients that make up a pharmaceutical ingredient formulation. Perhaps,
it is the limiting step of product development and requires special attention throughout the industry involved in
this process. Therefore, it is necessary to apply the philosophy of quality by design, once it must plan, eliminate
or diminish interference and expenditure of resources [75].Therefore, when choosing a drug molecule to
develop new, safe and effective therapeutic alternatives,
it is important to gather information regarding the intrinsic characteristics of the molecule.Regarding
HCQS, Semeniuk and coworkers (2008) [4] demonstrated by techniques of crystallography and X-ray
diffraction, the crystal structure projection and intermolecular interactions of the monocrystal drug. According
to the study, each of the nitrogen atoms of the free base is a proton donor in intermolecular hydrogen bonds with

57

Clinical, pharmacokinetic and technological

the oxygen atoms of the sulfate anion. Moreover, because it is a sulfate salt, the formation of "supermolecules"
containing two cations and two anions can be observed, it is the free base of the drug and sulfate,
respectively. The study also compared the presence of the hydroxyl group in relation to its absence in
chloroquine
phosphate, where it has been suggested that the addition of another proton donor appears to promote
significant changes in the conformation of the molecule, however it increases the interaction with the
transmembrane receptor of the parasite (putative receptor) [76].On the salt formation, it is suggested that
synthesis of the sulfate salt is due to two parameters: solubility and melting point. The solubility of HCQS is
2,61.10-2 g/L, therefore classified as practically insoluble, according to the Brazilian Pharmacopoeia 5 th Edition
[77-79]. Very little is known about the stability and other information of HCQS in the field of pharmaceutical
technology. Officially, the British and American official compendia dont mention stability issues of HCQ such
as impurities and degradation products [80-81]. However, some authors have demonstrated the existence of
substances correlated from forced degradation studies.Tnnesen and collaborators (1988) [49] irradiated drug
solutions in water and isopropanol using preparative HPLC to isolate impurities. Samples followed, then the
analysis of NMR and mass spectrometer (MS) (isobutane chemical ionization). After only 5 hours of exposure,
the irradiated sample in isopropanol originated a chromatogram with four different impurities (IMP): IMP -2
(desethyl-HCQ), IMP-3 (N-desidroxiethyl-HCQ), IMP-4 (dimer of HCQ) and IMP-5 (deaminated-HCQ).
Later, Dongre and colleagues (2009) [82] carried out the identification and characterization of two
impurities related to the synthesis of the drug: the already mentioned IMP-2 and the novel one, the IMP-1 (Nquaternary HCQ). The identification and characterization of these substances was possible only by the use of
hyphenated techniques, such as LCMS-IT analysis followed by ESI-TOF and NMR (1H, 13C, DEPT and 2D) of
the
drug
and
isolated
impurities.More
recently,
Saini
and
Gulshan
(2013)
[83]
in short communication conducted the first study of forced degradation. The work proved to be successful in its
objective: to conduct the study of forced degradation, identifying all possible degradation products coming from
the different reactions of hydrolysis, oxidation, photolysis, and dry heat.Virtually all proposed conditions
showed stability of the drug, except the alkaline solution maintained under photolysis conditions. This solution
showed six degradation products (Fig. 1): four unpublished (I-IV) and two products with similar structure to
known impurities. The authors highlighted that the degradation products III-VI have intact the chromophore
group of the drug, since they retain the same pattern of the UV absorption spectrum. While, the degradation
products I and II show changes in the chromophore group Benzenoid. Thus, it was possible to characterize most
of the degradation products, except VI, which offered little in the mass spectrum results; III is already known
impurity, while I, II, IV and V were new degradation products elucidated by the authors.

Figure 1. Structures of HCQ, its impurities and degradation products (adapted from Saini and Bansal, 2013[83]).

58

Clinical, pharmacokinetic and technological

To obtain these results, the authors were offered the following characterization techniques: LC-PDA, +
ESI-MSn and LCMS-TOF; to elucidate the most probable route of degradation level (Fig. 2), as well as the
intrinsic characteristics of the drug stability.

Figure 2. Probable degradative route of HCQS.

The use of HCQS requires attention, because it is commercialized in the racemic form. The use of
racemic mixtures can contribute to toxicity or adverse drug effects, particularly when they are associated with
pharmacologically inactive or less active isomers. Practical example: thalidomide. Just one of its enantiomers is
able to promote sedation. When metabolized, the molecule undergoes racemization in vivo and just the most
toxic form is absorbed, responsible for the notorious birth defects [84-87]. Then, it is necessary to better clarify
the physico-chemical properties and stereoselective profiles - kinetic and dynamic of chiral drugs. Hence,
drugs that are already marketed as racemates have been studied to assess whether there is an advantage in
producing its pure enantiomer [84]; [86].
Therefore, the development of analytical methods capable of separating isomers in known
concentrations, in biological or pharmaceutical preparations, has become an essential requirement in quality
control and pharmacokinetic [86]; [88].Most studies of enantiomeric separation, techniques have been
developed using capillary electrophoresis and, more commonly, high performance liquid chromatography
(HPLC). For the success of HPLC, three procedures may be used: chiral derivatization, addition of chiral
additive to the mobile phase and use of chiral stationary phase (chiral column) [49]; [82]; [89-91].The chiral
derivatization involves a reaction of the mixture of enantiomers with a chiral derivatizing agent,
enantiomerically pure, to form two diastereomeric derivatives. Then, the diastereomers may be separated using
HPLC on eluting mode in the reversed phase or normal phase. In the case of HCQS, there are three sites of
formation of diastereomers: aromatic nitrogen, tertiary aliphatic nitrogen and hydroxyl group; as shown in Fig. 3
[82]; [86].

59

Clinical, pharmacokinetic and technological

Figure 3. Sites of formation of isolated diastereomers of HCQS.

The use of (+)-di-O-acetyl-L-tartaric acid anhydride (DATAAN) as the derivatization reagent was first
reported by Lindner and collaborators (1991) [92]. The work includes the use of DATAAN for the separation of
the enantiomers of various -adrenoceptor antagonists, including propranolol. Instead of the amino function,
DATAAN preferentially reacts with the hydroxyl group of -blockers to form esters, which justifies its use for
HCQS. Further studies demonstrate the use of other derivatizing agents, such as S-(+)-1-(1-naphthyl)ethyl
isocyanate and S-(+)-1-(1-phenyl)ethyl isocyanate [64].
In addition chiral additive - High enantiomeric separation mechanism -, one chiral selector is added to
the mobile phase. Transient diastereomeric complexes formed between the analyte and the mobile phase plus
the chiral selector may also be separated by the HPLC on elution mode in the reversed phase or normal phase
[86]; [93].The method using chiral column - third and final method presented here - seems to be the most
applied. The procedure involves using a chiral selector chemically bound to the stationary phase. Recent
methods use the 1-acid glycoprotein as the bound agent. Such a system is able to interact with the two
enantiomers of the analyte, forming transient diastereomeric complexes through hydrogen bonds, -
interactions, inclusion complexes and steric avoidance, leading consecutively to enantioseparation [86]; [94-97].
The large standoff encountered in the development of analytical methods for enantioseparation lies in
obtaining the isolated compounds with high purity. Chemical reference substances of drugs, metabolites and
their isomers are expensive or nonexistent. Therefore, some studies use internal standards. Ofori-Adjei and
collaborators (1986) [98] incubated (R) and (S) isomers of chloroquine separately with human liver
microsomes, in vitro, producing optically pure enantiomers of metabolites, subsequently used as standard.In the
case of HCQ, the racemic mixture contains two isomers: (-)-(R)-HCQ and (+)-(S)-HCQ. The biological
metabolism of the drug promotes the formation of three main active metabolites: Desethylchloroquine (DCQ)
desethylhydroxychloroquine (DHCQ) and bisdesethylchloroquine (BDCQ), all chiral compounds [70]; [72];
[99].
The methods described for the enantioselective analysis of HCQ and its main metabolites using HPLC
techniques include two-step analysis. The analytes include: pharmaceutical preparations, urine, plasma and
whole blood.Another way to assess the stability of a drug is thermodegradation. Often it can be measured by
thermogravimetric analysis, a technique capable of assessing the interdependence of the mass variation which
occurs in the sample - gain or loss - as a function of time (at a given constant temperature) or temperature
[100]. However, there is no currently information in the literature about thermal behavior and stability of the
solid form of HCQ sulfate.

V.

CONCLUSION

Despite the classical indication as an antimalarial drug, HCQS is presenting a diversity of clinical
evidence that awaken the search for a better understanding of their biological properties. Among the activities
reported in the literature, there are indications for the treatment of autoimmune diseases such as lupus and RA,
where the drug is already an established alternative therapy. Reports also reveal the effectiveness of HCQS in
different diseases from those present in rheumatic conditions, such as improved insulin sensitivity, ability to
reduce thromboembolic events, reduction of lipid levels and treatment of infection by HIV.

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Clinical, pharmacokinetic and technological

The found evidences reinforce the need for special care in the treatment management in long term with
HCQS, since the ocular and cutaneous adverse effects, possible phototoxic reactions, can cause irreversible
damage. The biological metabolism of HCQS promotes the formation of three active and chiral metabolites, a
fact that encourages the development of studies that evaluate the kinetic behavior of these metabolites and the
intrinsic characteristics of the drug, because the literature has been reported in a preliminary form.

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